In technical fields relating to chemical formulation of compounds, such as, but not limited to the fields of pharmaceutical and agrochemical research and development, it is almost always necessary to evaluate the general suitability of a newly developed drug candidate prior to launching into full development. Such an evaluation of the general suitability or, in the field of pharmaceutical development, drug-ability of such chemical compounds typically includes solubility studies of the compound in various solvents as well as solubility profiles at various pH values. However, carrying out such studies for a great many compounds can be problematic and resource-intensive. At the earlier stages of the drug discovery process, in particular, the solubility measurements are generally performed for a large variety of compounds. Furthermore, many of these compounds are only available in limited quantities, either due to difficulties in manufacturing larger quantities, handling all but the smallest sized samples, or simply because the cost of producing or handling larger quantities of the compounds is not feasible.
However, simply bypassing the solubility studies is also not a viable option for product development as selection of an otherwise suitable candidate compound that does not have a suitable solubility profile can cause significant problems. Indeed, insoluble or poorly soluble compounds often prove difficult to develop into drugs. Even with significant motivation, the development of low-solubility drugs is more time-consuming and expensive than for a compound with otherwise more suitable properties.
Traditionally, “equilibrium” solubility has been determined by agitating or shaking the compound with the solvent of choice for at least 24 hours or until no more of the compound will dissolve, then filtering, and determining the concentration of dissolved compound by a suitable analytical assay. These analytical assays had to be calibrated, a process which includes preparation of at least several solutions of the known varied concentrations of the compound (standard solutions), and establishing a quantitative relationship between a measurable analytical signal and the compound concentration. This approach is inappropriate in a modern drug discovery setting. The throughput, number of unknown samples that can be determined in a given amount of time or using a given quantity of resources, such as machines, personnel, samples, and the like, is not high enough to meet the required demand to analyze a great number of potential lead compounds. For example, determination of the mass of samples and/or standards presents too restrictive a checkpoint in the process for maintaining the high throughput desired as the process of weighing hundreds (or thousands) of solid samples in submilligram quantities.
Therefore, to alleviate the perceived hindrances to high throughput analysis of compounds, those of skill in the art have sought to develop improved methods for determining solubility of compounds. One of these methods is based on measuring turbidity of an aqueous media after adding a fixed amount of solution of a compound in dimethyl sulfoxide (DMSO) by using laser nephelometry (Bevan & Lloyd, “A high-throughput screening method for the determination of aqueous drug solubility using laser nephelometry in microtiter plates,” Anal. Chem. 72, 1781-1787 (2000)). However, this method is limited in that it does not allow one to measure solubility of compounds in pure aqueous media without DMSO. Another method suggested in the literature is based on measuring the vapor pressure depression for a solution of the compound at saturation (Parikh et al., “Rapid solubility determination using vapor-phase osmometry,” J. Biomol. Screen. 4, 315-318 (1999)). However, this method is limited to use for measuring the solubility of nonionic compounds with rather good solubility in pure water. Furthermore, it cannot be used for poorly soluble compounds, and its use for solutions of ionic compounds in buffer or salt solutions is considered questionable (Parikh et al., “Rapid solubility determination using vapor-phase osmometry,” J. Biomol. Screen. 4, 315-318 (1999)).
Additional methods, such as those available from pIon Inc., are based on producing experimental samples by mixing DMSO solution of a compound of interest with a given aqueous solvent, incubation of the mixture for a fixed period of time, removing the precipitant formed by filtration, and assaying the compound concentration by measuring the optical absorbance of the filtrate at the maximum wavelength specific for the compound. However, differences between these assays and those to determine the concentration of compounds to use or to determine the optical absorbance values to use in these additional methods give rise to difficulties. For example, a concentration assay (similar to those used to determine solubility, but without incubation and filtration) is performed in a separately prepared mixture of the same DMSO solution of the compound with the same aqueous solvent but using a higher ratio of DMSO to aqueous solvent in the mixture. Under suitable conditions, the higher DMSO/aqueous solvent ratio is such that the compound is not precipitated out of solution. Correspondingly, the measurement of absorbance is taken to indicate the absorbance for a given quantity of the compound. This measurement when all the compound is solubilized, or rather, sets of these measurements are used as reference points to generate a relationship between the measured absorbance value and the quantity of compound. In determine the relationship, essentially a standard curve as is known in the art, the linearity of the optical absorbance vs. concentration over the used DMSO/aqueous solvent ratio range is assumed. Comparison of the standard curve and the measured value determined from the experimental sample, following incubation and filtration, is used to calculate/determine the concentration of the compound in the experimental sample.
In general, procedures like those available pIon have additional disadvantages which limit their practical application under some circumstances. In particular, the technique as outlined above requires that it be possible to determine the initial concentration of a compound under study in DMSO or in a DMSO-containing solvent. Further, this method is limited to compounds with chromophoric groups, such that they can be detected by absorbance measurements. Procedures that do not require that the compounds to be amenable to their initial concentration being determined in DMSO or a similar solvent or that do not require that the compounds have easily detectable or commonly used chromophoric groups would be a significant advancement of the technology to test the solubility of compounds.
The present invention provides new methods for determining the solubility of compounds of interest in solvents of interest. In specific embodiments, this includes methods that allow measurement of the solubility of organic, inorganic and organo-metallic compounds, particularly of compounds related to pharmaceutical research and development, in aqueous media with or without organic solvent. Further, the provided method is not necessarily limited by the ionization state of the compound or by the presence of inorganic salts and/or buffer salts in the media. Further, the provided methods are adaptable for high-throughput automated measurements. These and other objectives of the invention will become apparent in view of the detailed description below.